Cellular interactions can be viewed as proceeding in two steps. Initially, an extracellular molecule binds to a specific receptor on a target cell, converting the dormant receptor to an active state. Subsequently, the receptor stimulates intracellular biochemical pathways leading to a cellular response, which may involve progression through the cell cycle, as well as changes in cellular gene expression, cytoskeletal architecture, protein trafficking, endocytosis, cell adhesion, migration, proliferation and differentiation, among others. An intracellular biochemical pathway which mediates some of these cellular responses involves members of the c-src family of protein tyrosine kinases, such as pp60c-src. Src tyrosine kinases transduce extracellular signals as diverse as responses to growth factors (for example, platelet derived growth factor (PDGF), epidermal growth factor (EGF)), antigens, cytokines, extracellular matrix molecules, among others. These extracellular signals give rise to a myriad of cellular responses, such as mitotic function, activation of Ras dependent pathways, phosphatidyl inositol 3-kinase activation and cytoskeletal reorganization.
The amino terminus of pp60c-src contains two motifs of approximately 100 and 60 amino acids in length named Src homology 2 and 3 domains (SH2, SH3), respectively. SH2 and SH3 domains have been identified in numerous signal transduction proteins (Pawson, T. and J. Schiessinger (1993) J. Curr. Bio. 3:434-442; Courtneidge et al. (1994) Trends Cell Biol. 4:345-347; Pawson, T. (1995) Nature 373: 573-580). These domains presumably function as modular units that interact with other signal transduction proteins. The importance of SH2 and SH3 domains in signal transduction is underscored by the identification of xe2x80x9cadapter proteinsxe2x80x9d, such as c-crk (Reichman et al., 1992), c-nck (Chou et al., 1992) and grb-2/ASH (Margolis et al., 1992; Matuokà et al., 1992), which lack a catalytic domain, and thus, appear to function as adaptors between membrane signaling and multiple downstream targets.
Proteins containing SH2 domains control biochemical pathways as diverse as phospholipid metabolism, tyrosine phosphorylation and dephosphorylation, activation of Ras-like GTPases, gene expression, protein trafficking and cytoskeletal architecture (Pawson, T. and J. Schlessinger (1993) J. Curr. Bio. 3:434-442). In vivo, SH2-containing proteins bind to phosphotyrosine (pTyr)-containing sites on activated receptors and cytoplasmic phosphoproteins (Anderson et al. (1990) Science 250:979-982; Matsuda et al. (1990) Science 248:1537-1539; Valius, M. and A. Kazlauskas (1993) Cell 73:321-334). Indeed, crystal structures of the SH2 domains show a pocket configuration of amino acids that interact directly with a phosphotyrosine residue of an associated protein. Based on the crystal structure, the amino acid residues adjacent to the residues in direct contact with the phosphotyrosine determine the specificity of the interaction (Waksman et al. (1993) J. Cell 72:779-790; Lee et al. (1994) Structure 2:423-438).
SH3 domains have been found in a number of proteins involved in tyrosine kinase signaling, but also in cytoskeletal components and subunits of the neutrophil cytochrome oxidase, among others (Drubin et al. (1990) Nature 343:288-290; Leto et al. (1990) Science 248:727-730). In contrast to SH2 domains which interact with phosphorylated tyrosine residues of an associated protein, phosphorylation does not appear to be necessary for a protein to interact with a SH3 domain. The first SH3 binding protein identified, 3bp-1, shows homology to rho GTPase activating protein (GAP) (Cicchetti et al., (1992) Science 257:803). C3G was initially identified as a GTP exchange factor for several G proteins, and was subsequently shown to have affinity for the SH3 domains of Crk and Grb-2 (Tanaka et al. (1994) Proc. Natl. Acad. Sci. USA 91:3443-3447). G proteins themselves may be the targets for the binding of SH3 containing proteins. As an illustration, the proline rich C-terminus of the brain specific form of dynamin binds to several SH3 domains including those found in pp60c-src and pp59c-fyn, but not pp58c-fgr (Gout et al., 1993; Seedorf et al. (1994) J. Biol. Chem. 269:16009-16014). Dynamin is a microtubule-associated GTPase that is involved in endocytosis (Takel et al., 1995; Hinshaw et al., 1995). The binding of a SH3 domain to dynamin results in an increase in intrinsic GTPase activity (Gout et al., 1993).
SH3-binding sites consist of proline-rich peptides of approximately 10 amino acids (Ren et al. (1993) Science 259:1157-1161; Yu et al. (1994) Cell 76:933-945), which bind to isolated SH3 domains with dissociation constants of 5-100 xcexcM (ref. 25). Recent structural and mutagenic analysis of peptide-SH3 complexes (Feng et al. (1994) Science 266:1241-1246; Lim et al. (1994) Nature 372:375-379; Musacchio et al. (1994) Nature Struct. Biol. 1:546-551; Wittekind et al. (1994) Biochemistry 33:13531-13539; Rickles et al. (1994) EMBO J. 13:5598-5604) shows that peptides associated with SH3 domains adopt a left-handed polyproline type II helix, with three residues per turn, as illustrated by a PXXP consensus sequence (P=Proline, X=any amino acid) that forms a polyproline type II helix (Yu et al. (1994) Cell 76:933-945). Solution and crystal structures of SH3 domains complexed with small peptides indicate a groove in the SH3 domain where the prolines of the PXXP helix are situated (Lim et al. (1994) Nature 372:375-379; Yu et al. (1994) Cell 76:933-945; Musacchio et al. (1994) Nature Struct. Biol. 1:546-551). Residues adjacent to the prolines also form contacts within the SH3 sequence and these interactions determine the specificity between a protein and a particular SH3 domain. For example, the arginine in xe2x80x9cRPLPXXPxe2x80x9d forms a salt bridge with aspartate at position 99 of pp60c-src. However the C-terminal arginine in the sequence xe2x80x9cAFAPPLPRRxe2x80x9d contacts the identical aspartate in pp60c-src, indicating that proteins may interact with SH3 domains in either a xe2x80x9cplusxe2x80x9d or xe2x80x9cminusxe2x80x9d orientation (named xe2x80x9cclass Ixe2x80x9d and xe2x80x9cclass IIxe2x80x9d binding, respectively; Yu et al. (1994) Science 258:1665; Lim et al. (1994) Nature 372:375-379).
Several proteins that interact with the SH3 domains of src-family kinases have been shown to be implicated in cellular growth. These include the regulatory subunit of phosphatidyl-inositol-3-kinase, p85 (Prasad et al. (1993) Proc. Natl. Acad. Sci. USA 91:2834-2838), SHC (Weng et al., 1994), and ras GTPase-activating protein (Briggs et al., 1995). Furthermore, mutants within the SH3 domains of the adapter proteins c-crk and grb-2 inhibit v-abl oncogenic activity presumably by acting as xe2x80x9cdominant negativexe2x80x9d signal transduction effectors (Tanaka et al. (1995) Proc. Natl. Acad Sci. USA 91:3443-3447).
Despite much progress in characterizing the signal trasnduction pathways involving SH3 domains, there is a great need for identifying novel mediators of these pathways, and in particular, binding proteins that interact with these SH3 domains. The identification of these novel molecules may provide for a detailed analysis of the amino acid contacts that determine the binding affinity and specificity of SH3 domains with an associated protein, which may in turn facilitate the development of therapeutic agents to be used in treating a diverse number of disorders.
The present invention is based, at least in part, on the discovery of nucleic acid molecules which encode a novel family of src SH3 binding proteins, referred to herein as xe2x80x9cdifferentiation enhancing factorsxe2x80x9d or xe2x80x9cDEF polypeptidesxe2x80x9d. The DEF molecules show a highly conserved N-terminal domain and divergent C-terminus. The N-terminal domain preferably includes several structural motifs such as at least one src SH3 consensus binding sequence, at least one, and preferably four ankyrin repeats, at least one zinc finger domain, at least one pleckstrin homology domain and at least one C2 domain. The C-terminal domain diverges between family members, and may include at least one, and preferably three, more preferably six copies of a proline-rich tandem repeat and an SH3 domain. In one embodiment, DEF molecules of the invention are cytoplasmic proteins which function as mediators of signal transduction pathways of, for example, SH3 domain containing molecules, thus mediating multiple events including gene expression, cytoskeletal architecture, protein trafficking and endocytosis, cell adhesion, migration, proliferation and differentiation. In a preferred embodiment, DEF molecules of the invention modulate the differentiation of precursor cells, e.g., adipose or neural precursor cells. The DEF molecules of the invention may therefor be useful in the treatment of disorders, for example, hyperplastic and neoplastic tissues.
In one aspect, the invention provides isolated nucleic acid molecules encoding a DEF polypeptide. Such nucleic acid molecules (e.g., cDNAs) have a nucleotide sequence encoding a DEF polypeptide or biologically active portions thereof, such as a polypeptide having one or more of the following characteristics: the ability to bind to an SH3 domain in an intra- or intermolecular interaction; the ability to dimerize with like molecules or other molecules; the ability to anchor cytoskeletal elements to the plasma membrane; the ability to modulate the activity of signal transduction molecules, e.g., kinase activity, e.g., p38 MAP kinase activity, or G protein activity, e.g., GTPase activity; the ability to synergize with the activity of peroxisome proliferator activated receptor xcex3 (PPARxcex3); the ability to induce expression of PPARxcex3; the ability to induce the terminal differentiation of a hyperproliferative cell; or the ability to induce adipogenesis or neurogenesis. In a preferred embodiment, the isolated nucleic acid molecule has a nucleotide sequence shown in FIG. 2, SEQ ID NO: 1; FIG. 13, (SEQ ID NO: 3 or SEQ ID NO: 5); FIG. 14, (SEQ ID NO: 6 or SEQ ID NO: 8); or FIG. 15, (SEQ ID NO: 9 or SEQ ID NO: 11), or a portion thereof such as the coding region of the nucleotide sequence of FIG. 2, SEQ ID NO: 1; FIG. 13, (SEQ ID NO: 3); FIG. 14, (SEQ ID NO: 6); or FIG. 15, (SEQ ID NO: 9). Other preferred nucleic acid molecules encode a protein having the amino acid sequence of FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10). Nucleic acid molecules derived from a mammalian, preferably, a human cell (e.g., a naturally-occurring nucleic acid molecule found in a mammalian brain or an adipocyte cell) which hybridize under stringent conditions to the nucleotide sequence shown in FIG. 2, SEQ ID NO: 1; FIG. 13, (SEQ ID NO: 3 or SEQ ID NO: 5); FIG. 14, (SEQ ID NO: 6 or SEQ ID NO: 8); or FIG. 15, (SEQ ID NO: 9 or SEQ ID NO: 11) are also within the scope of the invention.
In another embodiment, the isolated nucleic acid molecule is a nucleotide sequence encoding a protein having an amino acid sequence which is at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95-99% overall amino acid sequence identity with an amino acid sequence shown in FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10). This invention further pertains to nucleic acid molecules which encode a protein which includes one or more of the following: at least one SH3 consensus binding sequence having an amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95-99% identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10); at least one ankyrin repeat, preferably two or three, and most preferably four ankyrin repeats, having an amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95-99% identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10); a zinc finger domain having an amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95-99% identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10); a pleckstrin homology domain having an amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95-99% identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10); and a C2 domain having an amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95-99% identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10). Further within the scope of this invention are nucleic acid molecules which encode a protein which includes a proline-rich repeat having an amino acid sequence at least 80%, preferably at least about 90%, more preferably at least about 95-99% identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2. This invention also encompasses nucleic acid molecules which encode a protein which includes an SH3 domain having an amino acid sequence at least about 80%, preferably at least about 90%, more preferably at least about 95-99% identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2 or FIG. 12, SEQ ID NO: 4 or SEQ ID NO: 7.
Nucleic acid molecules encoding proteins which include one or more of the following: at least one SH3 consensus binding sequence having an amino acid sequence at least about 60% (preferably at least about 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2, or FIG. 12, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10; at least one ankyrin repeat having an amino acid sequence at least about 60% (preferably at least about 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2, or FIG. 12, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10, a zinc finger domain having an amino acid sequence at least about 60% (preferably at least about 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2 or FIG. 12, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10, a pleckstrin homology domain having an amino acid sequence at least 60% (preferably at least about 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a C2 domain having an amino acid sequence at least about 60% (preferably at least about 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2; or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10); a proline-rich repeat having an amino acid sequence at least about 60% (preferably at least about 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2, and an SH3 domain having an amino acid sequence at least about 60% (preferably at least about 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2 or FIG. 12, SEQ ID NO: 4 or SEQ ID NO: 7, are also within the scope of this invention.
Another aspect of this invention pertains to nucleic acid molecules encoding a DEF polypeptide fusion protein which includes a nucleotide sequence encoding a first peptide having an amino acid sequence at least about 80% (preferably at least about 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3, SEQ ID NO: 2 or FIG. 12, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10, and a nucleic sequence encoding a second peptide corresponding to a moiety that facilitates detection or purification or alters the solubility of this fusion protein, such as glutathione-S-transferase, or an enzymatic activity such as alkaline phosphatase, or an epitope tag.
In another embodiment, the isolated nucleic acid molecule is a nucleotide sequence encoding a polypeptide fragment of at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850-1125 amino acid residues in length, preferably at least about 5-250 amino acid residues in length, and more preferably at least about 10-200 amino acid residues in length corresponding to a protein having at least about 80% the amino acid sequence shown in FIG. 3, (SEQ ID NO: 2) or FIG. 12, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10. In a preferred embodiment, the polypeptide fragment has a DEF activity, e.g., induces adipogenesis or neurogenesis.
Moreover, given the disclosure herein of a DEF polypeptide-encoding cDNA sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 11), antisense nucleic acid molecules (i.e, molecules which are complimentary to the coding strand of the DEF polypeptide cDNA sequence) are also provided by the invention. Accordingly, the DEF nucleic acid molecule can be non-coding, (e.g., probe, antisense or ribozyme molecules) or can encode a functional DEF polypeptide (e.g., a polypeptide which specifically modulates, e.g., by acting as either an agonist or antagonist, at least one biological activity of the DEF polypeptide). In a preferred embodiment, a DEF nucleic acid molecule includes the coding region of FIG. 1, (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9).
Furthermore, in certain preferred embodiments, the subject DEF nucleic acids will include a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the DEF gene sequences. Such regulatory sequences can be used to render the DEF gene sequences suitable for use as an expression vector. This invention also encompasses cells transfected with said expression vector whether prokaryotic or eukaryotic and a method for producing DEF proteins by employing the expression vectors.
Accordingly, another aspect of the invention pertains to recombinant expression vectors containing the nucleic acid molecules of the invention and host cells into which such recombinant expression vectors have been introduced. In one embodiment, such a host cell is used to produce DEF polypeptide by culturing the host cell in a suitable medium. If desired, DEF polypeptide can be then isolated from the medium or the host cell.
Still another aspect of the invention pertains to isolated DEF polypeptides and active fragments thereof, such as peptides having an activity of a DEF polypeptide (e.g., at least one biological acitivity of DEF polypeptide, such as the ability to bind to a src SH3 domain, the ability to induce PPARxcex3 expression; or the ability to induce the terminal differentiation of a cell, e.g., an adipose or a neural precursor cell, e.g., a transformed adipose or a neural precursor cell). The invention also provides an isolated preparation of a DEF polypeptide. In preferred embodiments, the DEF polypeptide comprises an amino acid sequence of FIG. 3, (SEQ ID NO: 2), or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10). In other embodiments, the isolated DEF polypeptide comprises an amino acid sequence at least 60% identical to an amino acid sequence of FIG. 3, (SEQ ID NO: 2) or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10) and, preferably has an activity of DEF polypeptide (e.g., at least one biological activity of DEF polypeptide). Preferably, the protein is at least about 70%, more preferably at least about 80%, even more preferably at least about 90% and most preferably at least about 95-99% identical to the amino acid sequence of FIG. 3, SEQ ID NO: 2 or FIG. 12, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10.
This invention also pertains to isolated polypeptides which include one or more of the following: a src SH3 consensus binding sequence having an amino acid sequence that is at least about 60% (preferably at least about 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), at least one ankyrin repeat, preferably two or three, and most preferably four ankyrin repeats, having an amino acid sequence that is at least 50% (preferably at least 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a zinc finger domain having an amino acid sequence that is at least about 50% (preferably at least about 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a pleckstrin homology domain having an amino acid sequence that is at least about 50% (preferably at least about 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2; or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a C2 domain having an amino acid sequence that is at least about 50% (preferably at least about 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2); or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a proline-rich tandem repeat having an amino acid sequence that is at least about 50% (preferably at least about 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4), and an SH3 domain having an amino acid sequence that is at least about 50% (preferably at least about 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4 or SEQ ID NO: 7).
The invention also provides for a DEF polypeptide comprising a first peptide having an amino acid sequence at least about 80% identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10) and a second peptide corresponding to a moiety that facilitates detection or purification or alters the solubility of this fusion protein, such as glutathione-S-transferase, or an enzymatic activity such as alkaline phosphatase, or an epitope tag.
Polypeptides comprising a polypeptide fragment of at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850-1125 amino acid residues in length, preferably at least about 5-250 amino acid residues in length, and more preferably at least about 10-220 amino acid residues in length, and most preferably at least about 200 amino acid residues corresponding to a protein having at least about 80% the amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10). In a preferred embodiment, the polypeptide fragment has a DEF activity, e.g., induces adipogenesis or neurogenesis.
Still another aspect of the invention pertains to isolated DEF polypeptide and active fragments thereof, such as polypeptides having an activity of a DEF polypeptide (e.g., at least one biological acitivity of DEF, such as the ability to bind to an SH3 domain in an intra- or intermolecular interaction, a polypeptide capable of dimerizing to like molecules or other molecules, a polypeptide capable of anchoring cytoskeletal elements to the plasma membrane, a polypeptide capable of modulating the activity of signal transduction molecules, e.g., kinase activity, e.g., p38 MAP kinase activity, or G protein activity, e.g., GTPase activity, a polypeptide capable of inducing PPARxcex3 expression, a polypeptide capable of inducing the terminal differentiation of a hyperproliferative cell, e.g., a transformed cell, e.g., a transformed adipose cell, or a polypeptide capable of inducing adipogenesis or neurogenesis).
The invention also provides an isolated preparation of a DEF protein. In a preferred embodiment, the isolated DEF protein comprises an amino acid sequence at least 70% identical to an amino acid sequence of FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10) and, preferably has an activity of DEF (e.g., at least one biological activity of DEF). Preferably, the protein is at least about 80%, more preferably at least about 90-95%, even more preferably at least about 96-98% and most preferably at least about 99% identical to the amino acid sequence of FIG. 3, (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10).
In another embodiment, the DEF protein comprises an amino acid sequence of FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10). This invention also pertains to isolated polypeptides which include a src SH3 consensus binding sequence having an amino acid sequence that is at least 80%, preferably at least about 85%, more preferably at least about 86-99% identical to a src SH3 consensus binding sequence shown in FIG. 3, (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), at least one ankyrin repeat, preferably two or three, and most preferably four ankyrin repeats, having having an amino acid sequence that is at least about 80%, preferably at least about 85%, more preferably at least about 86-99% identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a zinc finger domain having an amino acid sequence that is at least about 80%, preferably at least about 85%, more preferably at least about 86-99% identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a pleckstrin homology domain having an amino acid sequence that is at least about 80%, preferably at least about 85%, more preferably at least about 86-99% identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2), or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a C2 domain having an amino acid sequence that is at least about 80%, preferably at least 85%, more preferably at least about 86-99% identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2), or FIG. 12, (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10), a proline-rich repeat having an amino acid sequence that is at least about 80%, preferably at least about 85%, more preferably at least about 86-99% identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4), and an SH3 domain having an amino acid sequence that is at least about 80%, preferably at least about 85%, more preferably at least about 86-99% identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4 or SEQ ID NO: 7).
The invention also provides for a DEF fusion protein comprising a first polypeptide having an amino acid sequence at least about 80% (preferably at least 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10) and a nucleotide sequence encoding a second polypeptide corresponding to a moiety that facilitates detection or purification or alters the solubility of the fusion protein, such as glutathione-S-transferase, or an enzymatic activity such as alkaline phosphatase, or an epitope tag. In preferred embodiments, the fusion protein comprises one or more of a src SH3 consensus binding sequence, an ankyrin repeat, a zinc finger domain, a PH domain, a C2 domain, a proline-rich repeat, or an SH3 domain of a DEF polypeptide.
Yet another aspect of the present invention features an immunogen comprising a DEF polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a DEF polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen includes an antigenic determinant, e.g. a unique determinant, from a protein having at least about 80%, preferably at least about 85%, more preferably at least about 87-99% identity with the amino acid sequence represented by one of FIG. 3 (SEQ ID NO: 2) or FIG. 12 (SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10).
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the DEF immunogen.
The invention also features transgenic non-human animals, e.g. mice, rats, rabbits, chickens, frogs or pigs, having a transgene, e.g., animals which include (and preferably express) a heterologous form of a DEF gene described herein, or which misexpress an endogenous DEF gene, e.g., an animal in which expression of one or more of the subject DEF proteins is disrupted. Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed DEF alleles or for use in drug screening.
The invention also provides probes and primers composed of substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least 6 consecutive nucleotides preferably at least 25 more preferably at least 40, 50 or at least 75 consecutive nucleotides of either sense or antisense sequences of FIG. 2 (SEQ ID NO:1), FIG. 13 (SEQ ID NO: 3 or SEQ ID NO: 5), FIG. 14 (SEQ ID NO: 6 or SEQ ID NO: 8), or FIG. 15 (SEQ ID NO: 9 or SEQ ID NO: 11) or natually occurring mutants thereof In preferred embodiments, an oligonucleotide of the present invention specifically detects a DEF nucleic acid relative to other nucleic acid in a sample. In yet another embodiment, the probe/primer further includes a label which is capable of being detected. The label group can be selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying dysfunctions associated with mis-expression of a DEF protein, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding a DEF protein; e.g. measuring a DEF mRNA level in a cell, or determining whether a genomic DEF gene has been mutated or deleted. These so-called xe2x80x9cprobes/primersxe2x80x9d of the invention can also be used as a part of xe2x80x9cantisensexe2x80x9d therapy which refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject DEF proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation. Preferably, the oligonucleotide is at least 12 nucleotides in length, although primers of 25, 40, 50, or 75 nucleotides in length are also encompassed.
Yet another aspect of the present invention concerns a method for modulating one or more of a cell by modulating a DEF biological activity, e.g., by potentiating or disrupting certain protein-protein interactions. In general, whether carried out in vivo, in vitro, or in situ, the method includes treating the cell with an effective amount of DEF or a DEF agent so as to alter, relative to the cell in the absence of treatment, at least one or more of (i) cellular gene expression, (ii) cell proliferation, (iii) cell differentiation, e.g., differentiation of adipose or neural precursor cells, (iv) signal transduction, (v) cytoskeletal architecture, (vi) protein trafficking, (vii) adhesion of a cell. Accordingly, the method can be carried out with DEF or a DEF agents such as peptide and peptidomimetics or other molecules identified in the drug screens devised herein which agonize or antagonize the effects of signaling from a DEF protein or ligand binding of a DEF protein, e.g., an intracellular target molecule, e.g., an SH3 domain-containing molecule, a G protein, e.g., GTPase protein, or a cytoskeleton molecule. Other DEF agents include antisense constructs for inhibiting expression of DEF proteins, and different domains of the DEF proteins that may act as dominant negative mutants of DEF proteins which competitively inhibit ligand interactions upstream and signal transduction downstream of a DEF protein.
In one embodiment, the subject method of modulating a DEF biological activity can be used in the treatment of hyperproliferative cell to modulate growth arrest and terminal differentiation of a cell. In a preferred embodiment, the modulation of DEF activity occurs in an adipocyte or neural cell, in order to modulate adipocyte or neuronal differentiation. In another embodiment, the subject method is used to modulate induce growth arrest and differentiation of a cancer cell.
In yet another aspect, the invention provides a drug screening assay for screening test compounds for modulators, e.g., inhibitors, or alternatively, potentiators, of an interaction between an SH3 domain-containing protein, e.g., a DEF molecule or a c-src protein tyrosine kinases, e.g., pp60c-src and a DEF polypeptide or a biologically active portion thereof, e.g., an SH3 binding domain. An exemplary method includes the following (a) forming a reaction mixture including: (i) a pp60c-src, (ii) a DEF or an SH3 binding domain, and (iii) a test compound; and (b) detecting interaction of the pp60c-src and DEF or an SH3 binding domain. A statistically significant change (potentiation or inhibition) in the interaction of the pp60c-src, and DEF or an SH3 binding domain in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of said interaction. The reaction mixture can be a cell-free protein preparation, e.g., a reconsituted protein mixture or a cell lysate, or it can be a recombinant cell including a heterologous nucleic acid recombinantly expressing the DEF polypeptide.
In another embodiment, an assay is provided for screening for modulators of an interaction between a DEF polypeptide or biologically active portions thereof, e.g., a src SH3 consensus binding sequence, an ankyrin repeat, a zinc finger domain, a PH domain, a C2 domain, a proline-rich repeat and an SH3 domain, with signaling molecules. As an illustrative embodiment, test compounds that modulate the interaction between a DEF polypeptide or an ankyrin repeat and a cytoskeletal molecule can be tested.
In preferred embodiments, the steps of the assay are repeated for a variegated library of at least 100 different test compounds, more preferably at least 103, 104 or 105 different test compounds. The test compound can be, e.g., a peptide, a nucleic acid, a small organic molecule, or natural product extract (or fraction thereof).
Another aspect of the present invention provides a method of determining if a subject, e.g. an animal patient, is at risk for a disorder characterized by unwanted biological activity of a DEF polypeptide. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a DEF protein; or (ii) the mis-expression of a DEF gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a DEF gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble DEF protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of a DEF gene or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the DEF gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the DEF gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a DEF protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the DEF protein.
Another aspect of the invention provides a method for inhibiting proliferation of a hyperproliferative cell, e.g., a neoplastic cell, comprising ectopically expressing DEF or a functional fragment thereof in a cell in order to induce differentiation of the cell. In one embodiment, ectopic expression of DEF in a precursor cell may result in the differentiation of a hyperproliferative cell, e.g., an adipocyte precursor cell, or a cells derived from an adipose tumor, e.g., lipomas, fibrolipomas, lipoblastomas, lipomatosis, hibernomas, hemangiomas and/or liposarcomas, into adipocytes. In other embodiments, activation of DEF may synergize with other signaling agents to augment the differentiated phenotype. Thus, DEF alone or in combination with other agents can be used for the treatment of, or prevention of a disorder characterized by aberrant cell growth.
For example, the subject method can be used in the treatment of disorders mediated by an aberrant activity of a PPARxcex3 receptor. The subject method can be used in treating disorders characterized by the aberrant activity of an adipocyte precursor cell, e.g., obesity.
As another example, the subject method can be used in the treatment of sarcomas, carcinomas and/or leukemias. Exemplary disorders for which the subject method may be used as part of a treatment regimen include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing""s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms"" tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
In certain embodiments, the subject method can be used to treat such disorders as carcinomas forming from tissue of the breast, prostate, kidney, bladder or colon.
In other embodiments, the subject method can be used to treat hyperplastic or neoplastic disorders arising in adipose tissue, such as adipose cell tumors, e.g., lipomas, fibrolipomas, lipoblastomas, lipomatosis, hibemomas, hemangiomas and/or liposarcomas.
In still other embodiments, the subject method can be used to treat hyperplastic or neoplastic disorders of the hematopoietic system, e.g., leukemic cancers. In a preferred embodiment, the subject is a mammal, e.g., a primate, e.g., a human.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.